Several analytical detectors are available for compound characterization, probing a wide range of physical or chemical properties. Although many detectors possess high selectivity, almost all detectors are responsive to certain interfering compounds, which can either skew, or totally prevent an accurate analysis of the species of interest. In general, due to the presence of multiple analytes in a sample mixture, as well as non-analyte interfering (matrix) components in the sample, a separation step is often required prior to analysis. Therefore, the effective coupling of the separation step to the detection scheme can dictate the ultimate success of the analysis.
Of the many separation platforms available, a widely used method is liquid chromatography (LC). Modern, commercially available LC systems are automated, have high-resolution and separation capacities, are both fast and reproducible, and are based on a variety of distinct separation mechanisms. Owing to their widespread application, many analytical detectors have been modified to couple with LC separations; flow-cell detectors for spectroscopic measurements represent a commonly used example. Many other detectors, such as nuclear magnetic resonance detectors, and mass spectrometry instruments, have also been directly coupled to LC separations.
A potential problem in directly coupling LC separations to analytical detectors lies in the dilution of analytes into the mobile phase (the carrier liquid) of the separation. Since the response of many detectors is concentration sensitive, analyte dilution into the mobile phase results in a loss of detection sensitivity. In addition, the carrier liquid may cause interference in the detector; a minor detector response to the carrier solvent can result in a significant background. Excess liquid solvent has, and continues to be a major concern with direct coupling of LC separations to mass spectrometry (MS) instruments, since these instruments must operate at high vacuum. In addition, if post-separation sample manipulation steps are required, the dilution of sample into carrier liquid may potentially interfere with subsequent workup. For these reasons, it is often necessary to enrich the analyte following a separation.
Analyte concentration or enrichment can be accomplished in one of two general ways: through selective capture or transmission of analytes from the mobile phase, or through selective elimination of the mobile phase. Solvent elimination is most easily achieved by evaporation of the more volatile solvent. Techniques for selected capture or transmission of analytes include molecular weight cutoff filters, dialysis, or capturing analyte onto a solid support. In most cases, these techniques have been demonstrated in an off-line fashion through collection and subsequent manipulation of discrete fractions from the LC separation. Since many fractions can potentially result from a single separation, off-line sample enrichment requires considerable effort. In addition, there is a high risk associated with analyte loss or contamination during the workup process. A method for the enrichment and collection of fractionated analytes in a continuous, automated fashion would allow for a more direct coupling of LC systems to analytical detection schemes, or to subsequent sample workup steps, and would therefore provide a more desirable system.
Several systems have been described in the art that directly incorporate an enrichment of separated components from LC systems for subsequent chemical analysis of the fractionated components. Several of these systems were designed to address the concerns associated with coupling LC to MS instruments. For example, the techniques of thermospray, electrospray, atmospheric pressure chemical ionization, and ionspray are all designed to reduce the solvent being transmitted to the high vacuum region of a mass spectrometer, while allowing the analytes to be transmitted. Similarly, momentum based particle jet separators and membrane separators attempt to selectively transmit analytes to the detector while reducing the amount of solvent (for a review of LC-MS coupling techniques, see for example Abian, J. J. Mass Spectrom. 34, 157-168 (1999)). Although relatively high flow rate couplings can be achieved using the aforementioned devices, the reduction of the solvent comes at the expense of reducing the transmission efficiency of analytes to the detector, with the analyte being spread out over a large spot on the detector or sample plate. Also, these devices are designed for direct coupling to MS instruments; sample collection for subsequent workup or analysis is therefore negated.
In other prior art, systems have been presented that rely on the recovery or collection of analytes from a flowing liquid stream by deposition onto a solid support, or into vials. In doing so, the analyte enrichment and solvent elimination is independent of the detection or subsequent sample workup. The topic of coupling LC separations to MALDI-MS has been reviewed (K. K. Murray, Mass Spectrom. Rev., 1997, 16, 283-299). LC systems have been designed to couple low flow rate separations (<10 μL/min), simplifying a direct coupling to various detectors. U.S. Pat. No. 6,175,112 to Karger et al., discloses a system for the deposition of LC effluent as a continuous track onto a moving sample support. However, such a system suffers from only minimal sample capacity (low flow rate), and consequently, has a lower detection dynamic range.
In order to enrich analytes from higher flow rate separations, various methods for deposition of the eluent onto a solid support have been developed. U.S. Pat. Nos. 4,823,009 and 4,843,243 to Biemann et al., disclose a device for solvent elimination and simultaneous capture of the separated analytes from LC effluent onto a solid, rotating disk. The effluent is heated, and nebulized by a sheath gas flow to achieve rapid evaporation of solvent, depositing the solid analytes on the rotating disk. Dedmezian et al. in U.S. Pat. No. 5,039,614 describe a similar design for coupling LC separation to MS from a solid support in which analytes are deposited on a heated rotating disc by evaporation of the solvent in a subatmospheric pressure environment. The temperature and pressure can be adjusted according to the solvent composition/flow rate. U.S. Pat. No. 4,740,298 to Andresen et al. describes a moving belt interface for coupling LC to MS. This device is also based on similar principles, using a heated nebulizer to deposit samples on a solid support.
Electrospray deposition of the eluent onto a solid support has also been reported, see for example R. C. Beavis, W. Ens, D. E. Main, and K. G. Standing, Anal. Chem. 1990, 62, 1259-1264.
U.S. Pat. No. 5,772,964 to Prevost et al, describes a capillary nozzle for use with liquid chromatographic effluent. An extended portion of the capillary nozzle (20 cm or longer) is directed through a heater which heats the capillary contents to above the boiling point of the solvent in order to evaporate solvent. An upstream nebulizer upstream of the nozzle injects a nebulizer gas into the liquid effluent from the liquid chromatograph. A sheath gas is used at the nozzle outlet to direct the output from the nozzle. This and other designs which use a temperature above the solvent boiling point, particularly when coupled with nebulizing gas, have the disadvantage of producing a conical spray at the nozzle outlet, which forms fine mists and spreads or scatters the analyte out over the collection target. While the use of a sheath gas can provide a concentric focus on the spray which results in smaller deposition spots, analyte loss cannot be prevented due to the difficulty of collecting all fine mists exited from the nozzle on the collection target.
U.S. Patent Application 2002/0092366 to Brock et al, discloses a method and apparatus for depositing liquid droplets from a liquid chromatograph onto a sample plate for mass spectrometry. A capillary nozzle is used to create a liquid droplet and an electric field is generated between the droplet and the plate to polarize the droplet such that it is pulled to the sample plate. This system has the disadvantage that the liquid sample is not concentrated prior to depositing on the sample plate and it can only handle low flow rates.
There remains a need for a device for enrichment of analytes from a flowing liquid stream operating at high flow rates (such as up to 500 μL/min), without the disadvantages of prior systems which use temperatures above the boiling point of the solvent, nebulizers, electric charges or aerosol formation, all of which result in spreading of the analyte. Such a device would be particularly useful as a generally applicable interface for coupling LC to analytical detection schemes such as MS, and particularly MALDI-MS, as well as for collection for subsequent sample workup. Analyte enrichment following LC separation will allow for maximal detection sensitivity and ease of sample workup with minimal analyte loss or contamination. Such a device would also be very useful for concentrating dilute analyte solution without separation such as in a flow injection system where the dilute sample is either continuously pumped to the interface or injected as a sample plug to a flow stream and carried to the interface.